1. Field of the Invention
This invention relates generally to the field of automated tissue staining apparatus, and in particular is a new method of introducing stains into tissue using electrophoresis.
2. Description of Related Art
Tissue staining is an ancient art by modern standards that goes back over one hundred years. Recently, efforts have been made to automate the procedure of applying different types of chemical and biochemical stains to tissue sections. Instruments that have been invented for this purpose include the Ventana Medical Systems' line of dual carousel-based instruments such as the 320, ES®, NexES®, BENCHMARK®, and the BENCHMARK® XT. Patents that describe these systems include U.S. Pat. Nos. 5,595,707, 5,654,199, 6,093,574, and 6,296,809, all of which are incorporated herein by reference in their entirety. Another type of automated stainer is the TechMate® line of stainers, described in U.S. Pat. Nos. 5,355,439 and 5,737,499, both of which are incorporated herein by reference in their entireties.
The rate of Immunohistochemical and in situ hybridization staining of microtome-sectioned tissue on a glass slide is limited by the speed at which the biomolecules of interest can diffuse into the tissue from an aqueous solution placed in contact with the tissue section. Intact tissue presents many barriers to diffusion such as the lipid bilayer membranes that enclose individual cells and organelles, and the effects of cross-linking that the fixation process generates. The protein antibody or DNA probe molecules of interest are relatively large, ranging in size from a few kilo Daltons to several hundred kilo Daltons, which causes them to diffuse slowly into solid tissue with typical times for sufficient diffusion being in the range of several minutes to a few hours. A typical incubation period is thirty minutes at 37 degrees centigrade.
The diffusion rate is driven by concentration gradient so the rate can be increased by increasing the concentration of the conjugate in the reagent. However, this has two detrimental effects. First, the conjugates are often very expensive, so increasing their concentration is wasteful and not economically viable. Second, the excessive amount of conjugate that is driven into the tissue, when high concentrations are used, gets trapped in the tissue, and cannot be rinsed out and causes high levels of background staining. This background staining is called non-specific staining and, in an informational sense, is just noise. In order to reduce the noise and increase the signal of specific staining, low concentrations of conjugate are used with long incubation times to allow the conjugate to find and bind to only the specific sites.
Electrophoresis is an electrochemical separation technology commonly applied to separate biological molecules on the basis of their charge-to-mass ratio. Generally, a gel slab is prepared from a suitable polymeric material such as polyacrylamide by adding water to it in sufficient amount to create a semi-solid gelatinous slab. This is the matrix used to both contain the sample to be separated, and transmit the electric current used to electromotively move the various charged molecules. The pH of the gel can be manipulated to charge a biomolecule that is otherwise uncharged, thereby giving it the prerequisite net charge so that it will move when a field is applied to it. When the gel has an electric field applied to it, the charged molecules will migrate through the gel towards their opposite pole, i.e., negatively charged biomolecules will move towards the positive pole, and vice versa. The process is very commonly used in the biological research field to separate complex mixtures, and is termed “PAGE” (Polyacrylamide gel electrophoresis). A related technology is capillary electrophoresis (“CE”), which is the same basic electrochemical separation performed in thin glass capillary lumens filled with an electrolytic solution.
There continues to be a need for faster introduction of biomolecules into tissue sections, and for lower amounts of non-specific background staining.